Compositions and methods for performing assays

ABSTRACT

The disclosure relates to compositions for use in assays, the compositions comprising at least one latent fluorophore including at least one enzyme-reactive quenching group and a conjugative group; and a support connectable to the latent fluorophore by the conjugative group. The disclosure further relates to methods of measuring the presence and/or concentration of an analyte, as well as methods of measuring the relative activity of at least two enzymes.

TECHNICAL FIELD

The present disclosure relates to compositions and methods forperforming assays. In various embodiments, the disclosure relates tolatent fluorophores linked to a support, and their use in assays.

BACKGROUND

Assays are commonly used for qualitatively assessing or quantitativelymeasuring the presence, amount, or functional activity of a targetentity (the analyte). The analyte may, in various assays, be a drug,biochemical substance, or biological cell. By way of example, certainmedical conditions may utilize an assay to screen for the presence of atarget entity, or measure the amount of the target entity, in bodilyfluids such as urine, saliva, or blood.

For example, phenylketonuria (PKU) is a genetic condition characterizedby the inability to metabolize the amino acid phenylalanine. As aresult, individuals exhibiting this condition must control their intakeof phenylalanine, and some use blood tests (assays) to monitor theamount of phenylalanine (the analyte) in their blood serum. Likewise,blood serum levels of phenylalanine may be used to screen for, ordiagnose, PKU.

Methods for laboratory quantitation of phenylalanine levels in seruminclude a variety of enzyme assays having colorimetric or fluorescencesignals. For example, latent fluorophores are compounds with intensefluorescence that is revealed by a user-designated chemical reaction.Latent fluorophores using a trimethyl lock mechanism to cloak or quenchfluorescence can be used to detect levels of phenylalanine by couplingthe reaction of phenylalanine dehydrogenase to a diaphorase-activatedtrimethyl lock quenched latent fluorophore. The fluorescence intensityof the sample may be substantially directly proportional to the amountof phenylalanine present. This technique can provide a broad dynamicrange, but requires several blood sample processing steps to improve thesignal-to-noise ratio, such as isolating the serum from whole blood anddeproteinizing the serum.

The above-mentioned processing steps require equipment and techniquesthat are not ideal for adaptation to at-home testing devices. At-homephenylalanine testing devices are desirable though, as they may allowfor more frequent and convenient monitoring of blood phenylalaninelevels.

There is, therefore, a need for at-home phenylalanine testing devicescapable of testing whole blood, and capable of supplying a measurementsignal having a broad dynamic range and a high signal-to-noise ratio.Accordingly, there is a need for assays that may be useful in suchat-home devices, compositions for use in the assays, and methods ofmeasuring the concentration of an analyte, such as phenylalanine, in asample, such as whole blood. More generally, there is a need forcompositions and methods of detecting and/or measuring an analyte in asample.

SUMMARY

The disclosure relates, in various embodiments, to compositions for usein assays. The compositions may, in various exemplary embodiments,comprise at least one latent fluorophore comprising at least oneenzyme-reactive quenching group and at least one conjugative group; anda support connectable to the latent fluorophore, for example by at leastone conjugative group.

Further embodiments of the disclosure relate to methods for measuringthe concentration and/or presence of an analyte in a sample. In variousexemplary embodiments, the methods comprise one or more steps chosenfrom:

a. providing a fluorophore composition comprising:

-   -   i. at least one enzyme-reactive latent fluorophore comprising at        least one enzyme-reactive quenching group and at least one        conjugative group, and    -   ii. a support connectable to the latent fluorophore by at least        one conjugative group;

b. providing a test sample to be analyzed and a reference sample to beanalyzed;

c. contacting the test sample with the latent fluorophore composition,at least one first unquenching enzyme capable of releasing theenzyme-reactive quenching group from the latent fluorophore, and atleast one second enzyme capable of reacting with the analyte;

d. contacting the reference sample with the latent fluorophorecomposition and the at least one first unquenching enzyme;

e. measuring the fluorescence signal of the test sample and thefluorescence signal of the reference sample; and

f. comparing the fluorescence signal of the test sample with thefluorescence signal of the reference sample.

Further embodiments of the disclosure relate to methods for measuringthe activity and/or presence of an enzyme in a test sample including ananalyte. In various exemplary embodiments, the methods comprise one ormore steps chosen from:

a. providing a fluorophore composition comprising:

-   -   i. at least one enzyme-reactive latent fluorophore comprising at        least one enzyme-reactive quenching group and at least one        conjugative group, and    -   ii. a support connectable to the latent fluorophore by at least        one conjugative group;

b. providing a test sample to be analyzed and a reference sample to beanalyzed, wherein the reference sample contains a known quantity of theanalyte;

c. contacting the test sample with the latent fluorophore composition,at least one first unquenching enzyme capable of releasing theenzyme-reactive quenching group from the latent fluorophore, and atleast one second enzyme capable of reacting with the analyte;

d. contacting the reference sample with the latent fluorophorecomposition and the at least one first unquenching enzyme;

e. measuring the fluorescence signal of the test sample and thefluorescence signal of the reference sample; and

f. comparing the fluorescence signal of the test sample with thefluorescence signal of the reference sample.

Further exemplary embodiments of the disclosure relate to methods formeasuring the activities of at least two enzymes in a sample, forexample a multiplexed assay. In various embodiments, the methodscomprise one or more steps chosen from:

a. providing a first fluorophore composition comprising:

-   -   i. at least one first enzyme-reactive latent fluorophore        including at least one first enzyme-reactive quenching group and        at least one conjugative group, and    -   ii. at least one support connectable to the at least one first        latent fluorophore by at least one conjugative group;

b. providing a second fluorophore composition comprising:

-   -   i. at least one second enzyme-reactive latent fluorophore        including at least one second enzyme-reactive quenching group        and at least one conjugative group, wherein the at least one        second enzyme-reactive latent fluorophore is different from said        first enzyme-reactive latent fluorophore in said first        fluorophore composition, and    -   ii. at least one support connectable to the at least one first        latent fluorophore by at least one conjugative group;

c. providing a test sample to be analyzed and a reference sample to beanalyzed;

d. contacting the test sample with the first and second latentfluorophore compositions, at least one first unquenching enzyme capableof releasing the enzyme-reactive quenching group from the first latentfluorophore, and at least one second unquenching enzyme capable ofreleasing the enzyme-reactive quenching group from the second latentfluorophore;

e. contacting the reference sample with the first and second latentfluorophore compositions;

f. measuring the fluorescence signals of the test sample and thefluorescence signals of the reference sample; and

g. comparing the fluorescence signals of the test sample with thefluorescence signals of the reference sample.

Yet a further exemplary embodiment of the disclosure relates to a kitcomprising a composition, where the composition comprises at least onelatent fluorophore comprising at least one enzyme-reactive quenchinggroup and at least one conjugative group, and at least one supportconnectible to the at least one latent fluorophore via at least oneconjugative group.

Additional objects and advantages will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of various embodimentsaccording to the disclosure.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only,and are not restrictive of the invention, as claimed.

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several exemplary embodiments ofthe disclosure and, together with the description, serve to explain theprinciples of the disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an exemplary schematic of a coupled assay for detecting ananalyte having a specific dehydrogenase according to an embodiment ofthe disclosure;

FIG. 2 is an exemplary schematic of a coupled assay for detectingphenylalanine according to an embodiment of the disclosure;

FIG. 3 is an exemplary schematic of a direct assay for detecting ananalyte;

FIG. 4 a is an exemplary reaction schematic showing a latent fluorescentcompound revealed by a trimethyl lock reaction according to anembodiment of the disclosure;

FIG. 4 b is an exemplary reaction schematic showing a latent fluorescentcompound revealed by a trimethyl lock reaction and connected to amicrosphere with a biotin-streptavidin linkage according to anembodiment of the disclosure;

FIG. 5 is an exemplary reaction schematic showing a latent fluorescentcompound revealed by a double trimethyl lock reaction according to anembodiment of the disclosure;

FIG. 6 is a flowchart showing the steps for a general dehydrogenasedifferential assay according to an exemplary embodiment of thedisclosure;

FIG. 7 is a flowchart showing the steps for a phenylalaninedehydrogenase differential assay according to an exemplary embodiment ofthe disclosure;

FIG. 8 is an exemplary reaction schematic showing a latent fluorescentbiotin-containing compound revealed by a trimethyl lock reaction andsubsequently linked to a streptavidin-coated microsphere, according toan embodiment of the disclosure;

FIG. 9 a is a flowchart showing the steps for a general enzymaticmultiplexed differential assay using fluorophores having differentemission properties, according to an exemplary embodiment of thedisclosure;

FIG. 9 b is a flowchart showing the steps for an enzymatic multiplexeddifferential assay using fluorescent microspheres having differentemission properties, according to an exemplary embodiment of thedisclosure;

FIG. 9 c is a flowchart showing the steps for an enzymatic multiplexeddifferential assay using fluorescent microspheres having differentsizes, according to an exemplary embodiment of the disclosure;

FIG. 10 a is a flowchart showing the steps for a general enzymaticmultiplexed differential screening assay using fluorophores havingdifferent emission properties, according to an exemplary embodiment ofthe disclosure;

FIG. 10 b is a flowchart showing the steps for a general enzymaticmultiplexed differential screening assay using fluorescent microsphereshaving different emission properties, according to an exemplaryembodiment of the disclosure; and

FIG. 10 c is a flowchart showing the steps for a general enzymaticmultiplexed differential screening assay using fluorescent microsphereshaving different sizes, according to an exemplary embodiment of thedisclosure.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The disclosure relates, in various exemplary embodiments, tocompositions for use in an assay. In various exemplary embodiments, themeasurement from the assay is used to detect levels of phenylalanine inwhole blood.

In various exemplary embodiments, the compositions comprise at least onelatent fluorophore and at least one support. In further exemplaryembodiments, the composition is an enzyme-reactive latent fluorophorecloaked or quenched by an enzyme-reactive group, and the latentfluorophore is linked to the support. In at least certain exemplaryembodiments, the enzyme-reactive quenching group may be released by atrimethyl lock mechanism, and the enzyme-reactive latent fluorophore maybe revealed or unquenched by an enzymatic reaction. The resultingfluorescence intensity of the sample may, in certain embodiments, beproportional to the amount of enzyme activity. In various embodiments,the support may be chosen from microspheres, microbeads, or beads.

In various exemplary embodiments, the assay using the compositioncomprising the at least one latent fluorophore and at least one supportmay exhibit a broad dynamic range and high signal-to-noise ratio,although it should be noted that a broad dynamic range and highsignal-to-noise ratio are not required.

In at least certain embodiments, the latent fluorophore is attached tothe support with a linking group. By way of non-limiting example, thelinker may be chosen from polyethylene glycol (“PEG”) linkers. Incertain embodiments, the latent fluorophore is attached to the supportwith at least one conjugate. In at least one embodiment, the conjugatemay be biotin, and the support may be coated with a suitablecomplementary functional group, for example streptavidin.

According to various embodiments of the disclosure, compositionscomprising the latent fluorophore may be contacted with at least oneenzyme. In various exemplary embodiments, the latent fluorophore isattached to the support prior to the enzymatic reaction; in otherexemplary embodiments, it is attached after the enzymatic reaction.

In at least certain embodiments according to the disclosure, thefluorescence of the composition used in the analyte assay can bedetermined by measuring bulk fluorescence. In various embodiments, thefluorescence is measured using flow cytometry or spatially modulatedfluorescence detection technology. However, it should be noted that anymethod of measuring fluorescence known to those skilled in the art maybe used according to various embodiments of the disclosure.

Latent Fluorophores

As used herein, the term “latent fluorophore,” and variations thereof,means a chemical compound capable of exhibiting fluorescence, which maybe revealed by a designated chemical reaction. As used herein, the terms“quench,” “quenched,” and variations thereof mean a suppression orconcealment of some, all, or substantially all fluorescence of afluorophore by one or more functional groups, referred to herein as“quenching groups.” In its quenched state, a latent fluorophore exhibitsno or substantially no fluorescence, or a decreased amount offluorescence compared to its unquenched state.

The quenching group may quench or partially quench a latent fluorophoreby various chemical or physical means. In certain embodiments, thequenching group completes a system of conjugated double bonds whenaltered and/or released from the latent fluorophore, thus allowingfluorescence. The quenching group may be altered and/or released fromthe latent fluorophore by various chemical or physical means. In certainembodiments, for example, the quenching group may be altered and/orreleased from the latent fluorophore by a predetermined chemicalreaction. In further exemplary embodiments, the predetermined chemicalreaction may be catalyzed by an enzyme and/or an enzyme cascade.Nonlimiting examples of enzymes capable of catalyzing the unquenching orunlocking of a latent fluorophore include diaphorase and pyruvateoxidase.

Among the latent fluorophores that may be used according to exemplaryembodiments of the disclosure include, but are not limited to,fluorophores quenched by a quenching group releasable by a trimethyllock mechanism (trimethyl lock fluorophores), and fluorophores quenchedby an enzyme-activated trimethyl lock mechanism, such asdiaphorase-activated trimethyl lock fluorophores.

Latent fluorophores that may be revealed, unquenched, or unlocked by anenzymatic reaction, referred to herein as “enzyme-reactive latentfluorophores,” comprise an enzyme-reactive quenching group that may bealtered and/or released from the latent fluorophore upon reaction withor in the presence of the unquenching enzyme. In certain embodiments,the enzyme-reactive quenching group is a trimethyl lock. The resultingfluorophore, an active fluorophore, exhibits fluorescence. Because thealteration and/or release of the enzyme-reactive group is typicallyirreversible, for example due to the relative thermodynamic stability ofthe unreacted and reacted quenching group, the fluorescence signaltypically remains throughout any subsequent assay steps such ascollection, rinsing, and measurement.

According to various exemplary embodiments of the disclosure,fluorophores may be chosen to have certain specific excitation andemission properties. By way of example, the fluorophore may be chosenfrom fluoresceins and rhodamines. In certain embodiments, thefluorophore may optionally include amine groups on either side of thefluorophore.

The enzyme reactive group may be any moiety having sensitivity and/orspecificity toward an unquenching enzyme. In certain exemplaryembodiments, the enzyme reactive quenching group activates a trimethyllock mechanism and is then released from the enzyme-reactive latentfluorophore. A trimethyl lock is a functional group including threemethyl groups in close proximity. Steric interactions between thesethree methyl groups promote a lactone reaction and liberation of aleaving group. The resulting cyclic hydrocoumarin group isthermodynamically favored, making the trimethyl lock mechanism wellsuited as a fluorophore quenching group. In various embodiments, theunquenching enzyme reacts directly with the trimethyl lock to cleave theenzyme reactive group from the enzyme-reactive latent fluorophore.

Exemplary and non-limiting trimethyl lock fluorophores useful in variousembodiments may be chosen from, for example, compounds represented byChemical Formula 1, Chemical Formula 2, Chemical Formula 3, and mixturesthereof:

According to various exemplary embodiments of the disclosure, a latentfluorophore quenched by more than one enzyme-reactive quenching group,for example the compound represented by Chemical Formula 3, may bechosen, for example to increase the difference in fluorescenceintensity. By way of non-limiting example, in an embodiment where acompound represented by Chemical Formula 3 is chosen, theenzyme-reactive latent fluorophore is quenched by two enzyme-reactivegroups. In that exemplary embodiment, the compound fluoresces at a firstlevel of intensity when the first enzyme-reactive group is released, andfluoresces at a higher level of intensity when the secondenzyme-reactive group is released.

In various exemplary embodiments, more than one quenching group may bechosen, where different quenching groups have different properties, forexample the ability to be altered and/or released by reaction withcertain enzymes, rates of reaction, and conditions for reaction. Incertain embodiments, multiple latent fluorophores having differentquenching groups, and thus having different properties, may be chosenfor use in multiplexed assays. In certain embodiments involvingdifferent quenching groups, different unlocking enzymes and/or varyingreaction conditions may optionally be used.

Conjugative Group

According to various embodiments of the disclosure, the latentfluorophores may be connected to a support by at least one linkinggroup, or conjugative group. As used herein, the terms “connected,”“connection,” and variations thereof, mean being held in a set orpredetermined spatial relationship, or a set or predetermined spatialrelationship, and the term “connectable” and variations thereof meanscapable of being held in a set or predetermined spatial relationship. Asused herein, the terms “linking group,” “linker,” “conjugative group,”“bioconjugative group,” and variations thereof, which may be usedinterchangeably, mean a functional group or agent capable, by itself orin the presence of a complementary group, of forming a connectionbetween the latent fluorophore and the support.

In one embodiment, the linking group is a bioconjugative groupcomprising a pyrrole-2,5-dione group.

It may, in various embodiments, be desirable to choose a conjugate thathas a complementary functional group to which it is attracted. By way ofnon-limiting example, according to one embodiment of the disclosure, thelinking group may be chosen from a biotin group or biotin-containingcompound. It is known that the biotin group preferentially binds to theprotein streptavidin to form a strong protein-ligand interaction, whichmay be desirable according to certain embodiments of the disclosure. Forexample, a latent fluorophore containing a biotin linking group willform a connection with a support coated with streptavidin.

According to various embodiments of the disclosure, the linking groupmay include a polymer or oligomer having a degree of polymerization orpolymerization number, n, which indicates the number of repeated monomerunits. The polymerization number may determine the length of the polymeror oligomer linker. The polymer or oligomer linker can be of anyappropriate length to allow an enzyme to bind the enzyme-reactivequenching group on the enzyme-reactive latent fluorophore by providing asuitable space between the enzyme reactive group and the support. If nis too large, the polymer or oligomer linker will be too long, and maybe prone to breakage or other instability. Also, if the polymer oroligomer linker is too long, the fluorophore may form undesirableaggregates. If the polymer or oligomer linker is too short, it may bedifficult for the enzyme to access the reactive group. The length of thepolymer or oligomer linker can be adjusted so that the enzyme reactivegroup is accessible to the enzyme.

Thus, in various embodiments, n is any positive number. By way ofexample, n may range up to about 200, such as up to about 150, up toabout 100, up to about 75, up to about 50, up to about 25, or up toabout 20, such as about 1 to about 100, about 1 to about 25, about 1 toabout 20, about 3 to about 100, about 4 to about 100, about 3 to about25, about 4 to about 25, or about 4 to about 20. For example, in atleast certain non-limiting embodiments, n ranges from about 1 to about25. In yet further exemplary embodiments, n ranges from about 3 to about20, or about 4 to about 100. A PEG linker having n ranging from about 4to about 100 may result in a spacer length of approximately 16 to 110 Å.

In certain embodiments, the polymer or oligomer may be water soluble,biocompatible and/or nonreactive. Biocompatability of the polymer oroligomer may, in at least certain embodiments, advantageously decreasethe likelihood of interactions between the linking group and the enzyme,although it is not required. In various exemplary embodiments, thelinking group comprises a PEG linker or a dextran. In yet furtherexemplary embodiments, the linking group comprises a PEG linkerrepresented by Chemical Formula 4:

H—[O—CH₂—CH₂]_(n)—OH  Chemical Formula 4

According to various exemplary embodiments, the at least one conjugativegroup chosen may lead to improved stability and/or resistance toenzymatic degradation, although it is not required.

In various exemplary embodiments, more than one conjugative group mayoptionally be chosen, where different conjugative groups have differentproperties. For example, multiple conjugative groups having differentproperties may be chosen for use in multiplexed assays. In certainembodiments using multiplexed arrays, different latent fluorophores maybe connected to one or more types of supports by the same conjugativegroup.

Support

Supports useful according to various embodiments of the disclosureinclude, by way of non-limiting example, microspheres, microbeads, andbeads, as well as any other support to which the compounds describedherein can be attached. As used herein, the terms “microsphere,”“microbead,” and “bead,” which may be used interchangeably herein todenote any particulate support, as well as variations thereof, mean aparticle having a size ranging from about 0.1 μm to about 1000 μm.

It may be possible to increase the range of sensitivity in an assay bymodifying properties of the support, and/or by choosing supports with aspecific property or set of properties. For example, microspheres havinglarger or smaller diameters may be chosen, depending on the sensitivitydesired. A high surface area of the support allows a large number offluorophores to be attached thereto, which is thought to increase theoperating range of the assay. As such, the number of latent fluorophoresconnected to the support, e.g. a microsphere, can be adjusted to achievea desired operating range.

Further, supports having different functionalities, coatings,fluorescence excitation/emission characteristics, and/or scatteringcharacteristics, may be chosen. One of skill in the art will be able toselect a support having the appropriate properties, depending on, forexample, the intended application and/or specific latent fluorophorecomprising at least one enzyme-reactive quenching group and at least oneconjugative group to be used.

In various exemplary embodiments, more than one support may be chosen,where different supports have different properties. For example,multiple supports having different properties may be chosen for use inmultiplexed assays. In certain embodiments, different latentfluorophores may be connected to supports for use in multiplexed arrays.

By way of non-limiting example, more than one microbead havingsubstantially the same properties constitutes a class or type ofmicrobeads. Different classes or types of microbeads may be chosen wherethe microbeads have a variety of functionalities, fluorophores, and/orcoatings. More than one class or type of microbeads measuredsimultaneously constitute a multiplexed array.

For example, SPHERO™ Blue Fluorescent Particle Kits (Spherotech, LakeForest, Ill.) are available with carboxyl functionality for covalentattachment of ligands. These microbeads are produced with up to 10different amounts of fluorescent dyes incorporated in them. Depending onthe diameter of the beads, they are sold in kits of 7 to 10 individualclasses of microbeads. The classes differ in fluorescence intensity inthe PE-Cy5, allophycocyanin (“APC”), and APC-Cy7 channels with minimalfluorescence in the fluorescein isothiocyanate (“FITC”) andR-phycoerythrin (“PE”) channels, allowing identification of the beads'class. By using enzyme-reactive specific latent fluorophores whichfluoresce in the FITC or PE channel (for example, rhodaminederivatives), the specific class of enzymatic activity can be identifiedby the fluorescence intensity of the bead in the PE-Cy5, APC, andAPC-Cy7 channels.

As a further non-limiting example, microspheres having differentfluorescence excitation/emission characteristics may be chosen. Examplesinclude SPHERO™ Fluorescent Particles (Spherotech, Lake Forest, Ill.)and Fluorescent Microspheres (Bangs Laboratories, Fishers, Ind.).

A wide variety of fluorescent particles ranging in size, spectralcharacteristic of fluorescence, fluorescence lifetime, fluorescenceintensity, and/or surface functional group are available. Classes ofparticles that differ in their spectral characteristic of fluorescenceemission can be chosen to identify specific enzymatic activity,enzyme-reactive specific latent fluorophores which fluoresce in the FITCor PE channel that is associated with that class.

Depending on the application, the available excitation sources, and theemission filter(s) used, a variety of combinations can be selected. Forexample, available Internet tools such as the interactive spectrumviewer provided by BD Biosciences, Franklin Lakes, N.J., or theinteractive spectrum viewer provided by Life Technologies Corporation,Carlsbad, Calif. can be used for dye selection. In at least certainembodiments, combinations may be chosen so that the emissioncharacteristics (e.g., emission spectra) are sufficiently different sothat a first emission spectrum (for example, from an enzyme-reactivelatent fluorophore) does not significantly overlap with second andsubsequent emission spectrum(a) (for example, fluorescent microspheresof type 1, type 2, and type 3).

By way of yet further non-limiting example, microspheres havingdifferent scattering characteristics may be chosen. In at least onexemplary embodiment, SPHERO™ Polystyrene Particles, Spherotech, LakeForest, Ill., may be used in multiplex assays.

A wide variety of particle sizes with surface functional groups areavailable. For example, by linking a specific enzyme-reactive latentfluorophore (enzyme 1) to a bead of a specific size (microsphere type 1)and a second specific enzyme-reactive latent fluorophore (enzyme 2) to abead of a different size (microsphere type 2), the specific enzymeactivity (fluorescence from the latent fluorophore) can be associatedwith the scattering properties of the microsphere. The size of the beadsmay be selected so that there is a sufficient difference in thescattering properties of the various types of microspheres so that eachcan be uniquely identified.

In yet further exemplary embodiments, supports may be chosen that arecoated. By way of example only, streptavidin-coated microspheres may beused in various embodiments of the disclosure. In yet further,non-limiting exemplary embodiments, other binding proteins, or eithercomponent of any conjugative linking pair, may be coated on the support.

Magnetic Support

In at least certain exemplary embodiments according to the disclosure,magnetic microspheres, microbeads, and beads may be chosen as thesupport for the latent fluorophore. As used here, the terms “magneticmicrosphere,” “magnetic microbead,” and “magnetic bead,” which may beused interchangeably, as well as variations thereof, mean a particleexhibiting magnetism. For the manipulation of beads, paramagnetism orsuperparamagnetism in the presence of an externally applied magneticfield is often used. Paramagnetic materials exhibit a positive change inmagnetic moment in the presence of an externally applied magnetic field.Superparamagnetic materials exhibit high levels of paramagnetism. Thehigh levels of paramagnetism exhibited in superparamagnetic materialsare due to microscale or nanoscale ordering of spin orientations, whichmay be lacking in other paramagnetic materials. In certain exemplaryembodiments, the support comprises a superparamagnetic material.

Sizes of commercially available magnetic beads typically range fromabout 0.1 μm to about 120 μm. They often contain a paramagneticmaterial, for example iron oxide, resulting in the beads' paramagneticproperties, and a biocompatible shell that can be functionalized. Theparamagnetic material may constitute the core of the particle or may bea shell-like coating on a core, for example made of polystyrene.However, it should be noted that magnetic supports useful according tothe disclosure are not required to have any particular size,functionality, and the like, such as found in commercially availableproducts.

Because of their embedded paramagnetic materials, such beads can bemanipulated by an external magnetic field that induces a non-negligiblemagnetic field in the bead. In particular, the beads can be movedthrough a solution, collected, dispersed, mixed and extracted in acontrolled manner by means of an applied external magnetic field.Paramagnetic beads have the advantage of a magnetic field that vanishesonce the external field vanishes, so that the beads do not influenceeach other in undesirable ways, for example cluster together. Methodsfor controlled sample preparation involving such magnetic beads areknown in the art.

Additionally, fluorophores can be embedded in the bead for detection oridentification (for example UMC3F COMPEL™ Magnetic COOH modified,fluorescent particles from Bangs Laboratories Inc.). Connecting afluorophore to a magnetic microsphere may allow concentration of thefluorescence signal after performing an enzymatic reaction in a largervolume. The technique can allow the enzyme assay to be performed incomplex solutions, for example whole blood. It also enables efficientrinsing of interfering compounds, components, and the like, prior todetecting the fluorescence signal. In addition, fluorophores connectedto magnetic microspheres can, in at least certain exemplary embodiments,enable automated sample preparation and analysis.

Methods of Preparing Latent Fluorophores Linked to a Support

Any known method for preparing latent fluorophores comprising at leastone enzyme-reactive group and at least one conjugative group may beused. It is well within the skill of those in the art to designappropriate chemical reactions in order to prepare a latent fluorophorehaving at least one enzyme-reactive group and at least one conjugativegroup.

An exemplary and non-limiting method of synthesizing a latentfluorophore represented by Chemical Formula 1, which method is presentedsolely for purposes of illustrating one optional embodiment, is shown inReaction Scheme 1.

Any known connection between a support and latent fluorophorescomprising at least one enzyme-reactive group and at least oneconjugative group is also contemplated to be within the scope of thedisclosure. By way of non-limiting example only, a latent fluorophoresuch as that of Chemical Formula 1 prepared according to Reaction Scheme1 may be linked to the microsphere with a PEG linker. For example, aheterobifunctional PEG may be chosen, such as thiol-PEG-Amine (e.g.HS-PEG-NH₂; Creative PEGworks, Winston Salem, N.C.). The thiol groupreacts with the maleimide group on Chemical Formula 1, the amine groupis reacted with a microsphere containing a carboxylic acid functionality(for example, Dynabeads® M-270 Carboxylic Acid (magnetic,nonfluorescent) or FluoSpheres® Carboxylate-Modified Microspheres(fluorescent, nonmagnetic), both Life Technologies Corporation,Carlsbad, Calif.; Polybead® Carboxylate Microspheres (nonfluorescent,nonmagnetic) or Fluoresbrite® BB Carboxylate Microspheres (fluorescent,nonmagnetic); both Polysciences, Inc., Warrington, Pa.)) followingactivation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) to yield the desired latent fluorophore linked to amicrosphere. Optionally, the amine group at the end of the PEG linker onthe latent fluorophore can be linked to a Tosylactivated microsphere(for example, Dynabeads® M-280 Tosylactivated; Life TechnologiesCorporation, Carlsbad, Calif.).

In yet a further non-limiting exemplary embodiment where the latentfluorophore is chosen from those of Chemical Formula 2, thiol-PEG-acidmay be chosen (e.g. HS-PEG-COON; Creative PEGworks, Winston Salem, N.C.)to link to the support. The thiol group is reacted with the maleimidegroup on Chemical Formula 2, the carboxylic acid group is reacted with1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) andcrosslinked to a microsphere containing a carboxylic acid functionality(for example, Dynabeads® M-270 Carboxylic Acid; Life TechnologiesCorporation, Carlsbad, Calif., or Polybead® Carboxylate Microspheres;Polysciences, Inc., Warrington, Pa.).

In yet a further exemplary embodiment for preparing latent fluorophoresand linking to a support, the latent fluorophores comprising at leastone enzyme-reactive group and at least one conjugative group may besynthesized with a biotin group as at least one conjugative group. Thelatent fluorophore-biotin compound may then be reacted withstreptavidin-coated microspheres, to yield a latent fluorophore linkedto a microsphere.

Further, any known method of linking latent fluorophores comprising atleast one enzyme-reactive group and at least one conjugative group to asupport is also contemplated to be within the scope of the disclosure.In certain exemplary embodiments, the latent fluorophores mayspontaneously connect to the support when both are placed in solution.In further exemplary embodiments, the solution may be stirred, heated,and/or cooled to facilitate the linkage between the fluorophore and thesupport. In yet further exemplary embodiments, additives, for examplesurfactants, promoting agents, enzymes and the like, may be added to thesolution to facilitate the linkage between the fluorophore and thesupport.

Methods of Using Latent Fluorophores Linked to a Support

As used herein, the term “measuring enzyme activity” and variationsthereof refers to a measure of the quantity of active enzyme, and/or ofthe quantity of analyte acted upon by the enzyme. As used herein, theterms “an enzyme reacting with the analyte,” “an enzyme acting upon theanalyte,” and variations thereof mean a chemical reaction catalyzed byan enzyme, wherein an analyte is chemically altered. In certainembodiments, the chemical reaction may be a dehydrogenation reaction.

The fluorophore compositions described according to various embodimentsof the disclosure may be useful in enzyme assays. For example, thefluorophore compositions may be useful for methods of measuringactivities of enzymes. In various embodiments, the fluorescence signalof the unlocked fluorophore may be proportional to the enzyme activityto be measured due to the quenching group being altered and/or releasedby the enzyme in question, and/or by a downstream enzyme in a series ofenzyme reactions, or enzyme cascade, including the enzyme in question.In certain embodiments, the enzyme activities of two or more enzymes maybe compared.

Further, the fluorophore compositions may be useful for detecting and/ormeasuring the concentration of analytes in a sample, for example whenthe enzyme activity of a known concentration of enzyme in the presenceof an unknown amount of analyte is compared with the enzyme activity ofa known concentration of enzyme in the presence of a known amount ofanalyte.

As used herein, a sample may be any substance, in particular a sample ofliquid substance, and in at least certain embodiments, may be a sampleof bodily fluids such as urine, saliva, or blood. The samples usedaccording to various methods described herein may be obtained orcollected by any technique known. A “test” sample and a “reference”sample may, in at least certain embodiments, be two samples from thesame source, e.g. two samples of blood taken from one blood collection,where the test sample and reference sample may be treated with thecompositions described herein in different manners.

Methods of Measuring the Activities of Enzymes

Enzymatic activity dependent on the presence of a particular substrateis known to be correlatable with the concentration of that substrate.Compositions prepared according to various embodiments of the disclosuremay permit detection of such enzymatic activity via enzymatic assay.Exemplary enzyme assays useful according to various embodiments include,but are not limited to, enzyme cascade assays, coupled assays and directassays.

As used herein, the terms “contact,” “contacting,” and variationsthereof mean bringing components into close enough proximity to allowfor a chemical or enzymatic reaction to occur between or among thecomponents. The contacting may be between or among any combination ofcomponents in any manner. In various non-limiting exemplary embodiments,the contacting between a sample and a fluorophore is performed byincluding or mixing a fluorophore in a liquid sample. In certainembodiments, the liquid sample may be stirred, sonicated, heated and/orcooled, and/or additives may be added to the liquid sample to facilitatethe contacting between the sample and the fluorophore.

As used herein, the terms “compare,” “comparing,” and variations thereofmean determining the relative quantitative and/or qualitativefluorescence signals of two or more samples, by any method, andincluding either human or machine/computer determination. Thefluorescence signals of the samples may be compared by any knownmethods. In one non-limiting exemplary embodiment, the fluorescencesignals of two samples may be compared by subtracting the quantitativesignal of one sample from the quantitative signal of the other sample.

Methods for measuring the activity and/or presence of an enzyme in atest sample including an analyte include one or more steps chosen from:

a. preparing a fluorophore composition comprising:

-   -   i. at least one enzyme-reactive latent fluorophore comprising at        least one enzyme-reactive quenching group and at least one        conjugative group, and    -   ii. a support connectable to the latent fluorophore by at least        one conjugative group;

b. providing a test sample to be analyzed and a reference sample to beanalyzed, wherein the reference sample contains a known quantity of theanalyte;

c. contacting the test sample with the latent fluorophore composition,at least one first unquenching enzyme capable of releasing theenzyme-reactive quenching group from the latent fluorophore, and atleast one second enzyme capable of reacting with the analyte;

d. contacting the reference sample with the latent fluorophorecomposition and the at least one first unquenching enzyme;

e. measuring the fluorescence signal of the test sample and thefluorescence signal of the reference sample; and

f. comparing the fluorescence signal of the test sample with thefluorescence signal of the reference sample.

In various embodiments of the method of measuring the activity of anenzyme, the first unquenching enzyme that unlocks the latent fluorophoreadded to the test sample may be the same as or different from the secondenzyme capable of reacting with the analyte. When the first unquenchingenzyme is the same as the second enzyme, this may be referred to as adirect assay. When the first unquenching enzyme is different from thesecond enzyme, this may be referred to as an enzyme cascade or coupledassay, and in various embodiments the first enzyme may be considereddownstream of the second enzyme because the first enzyme uses aby-product of the reaction between the second enzyme and the analyte inorder to unquench the quenching group.

By way of non-limiting example, in one embodiment the first unquenchingenzyme that unlocks the latent fluorophore added to the test sample isthe same as the first unquenching enzyme added to the reference sample.When the same enzyme is added to both samples, the resulting differencein fluorescence may be attributed to the activity of the enzyme on theanalyte, when the only difference between the test sample and thereference sample is the quantity of analyte. In certain exemplaryembodiments, the reference sample is free or substantially free of theanalyte. In contrast, in a non-limiting embodiment where the test sampleincludes a different unquenching enzyme than the unquenching enzyme inthe reference sample, a difference in fluorescence signal between thetwo samples may be at least partially attributable to the difference inunquenching activity and/or rate between different unquenching enzymes.

Methods for measuring activities of two or more enzymes in a sample, forexample in a multiplex assay, include one or more steps chosen from:

a. providing a first fluorophore composition comprising:

-   -   i. at least one first enzyme-reactive latent fluorophore        comprising at least one first enzyme-reactive quenching group        and at least one conjugative group, and    -   ii. at least one support connectable to the at least one first        latent fluorophore by at least one conjugative group;

b. providing a second fluorophore composition comprising:

-   -   i. at least one second enzyme-reactive latent fluorophore        comprising at least one second enzyme-reactive quenching group        and at least one conjugative group, wherein the at least one        second enzyme-reactive latent fluorophore is different from said        first enzyme-reactive latent fluorophore in said first        fluorophore composition, and    -   ii. at least one support connectable to the at least one first        latent fluorophore by at least one conjugative group;

c. providing a test sample to be analyzed and a reference sample to beanalyzed;

d. contacting the test sample with the first and second latentfluorophore compositions, at least one first unquenching enzyme capableof releasing the enzyme-reactive quenching group from the first latentfluorophore, and at least one second unquenching enzyme capable ofreleasing the enzyme-reactive quenching group from the second latentfluorophore;

e. contacting the reference sample with the first and second latentfluorophore compositions;

f. measuring the fluorescence signals of the test sample and thefluorescence signals of the reference sample; and

g. comparing the fluorescence signals of the test sample with thefluorescence signals of the reference sample.

In various embodiments of the method of the multiplex assay above, thefirst unquenching enzyme that unlocks the latent fluorophore added tothe test sample may be the same as or different from the first enzymeadded to the reference sample. When the first unquenching enzyme is thesame as the first enzyme, this may be referred to as a multiplex directassay. When the first unquenching enzyme is different from the firstenzyme, this may be referred to as a first enzyme cascade, at in variousembodiments the first unquenching enzyme may be considered downstream ofthe first enzyme because the first unquenching enzyme uses a by-productof the reaction between the first enzyme and the analyte in order tounquench the first quenching group. An enzyme cascade including twoenzymes, one that reacts with the analyte and the other that unquenchesthe latent fluorophore, may be referred to as a coupled assay.

By way of example, in one non-limiting embodiment, the first quenchinggroup is different from the second quenching group. For a multiplexdirect assay, wherein the analyte-active enzyme is the same as theunquenching enzyme, the first and second quenching groups are unlockedby different unquenching enzymes; otherwise the difference influorescence signal could not be correlated with the activity of aparticular enzyme on a particular analyte. For a multiplex enzymecascade assay, wherein an enzyme having activity in the presence of ananalyte is part of an enzyme cascade that includes a downstreamunquenching enzyme, the first and second quenching groups are unlockedby different unquenching enzymes; otherwise the difference influorescence signal could not be correlated with the activity of aparticular enzyme cascade on a particular analyte.

In various embodiments, the first fluorophore has a first emissionspectrum that may be the same as or different from a second emissionspectrum of the second fluorophore. When the first and second emissionspectra have at least one different property, for example peak emissionwavelength, peak absorption wavelength, and/or fluorescence lifetime,the first and second fluorescence signals from the first and secondfluorophores, respectively, may be distinguished.

Alternatively, when the first and second emission spectra are the same,the first fluorophore may be distinguished from the second fluorophoreby employing other techniques. In various embodiments, one suchtechnique includes physically separating the first fluorophore from thesecond fluorophore, for example by linking at least one of thefluorophores to a support and physically separating the support, forexample by spatial concentration using an external magnetic, electric oracoustic field, or filtering; and/or linking the first fluorophore to afirst support and the second fluorophore to the second support, andsubsequently distinguishing the first support and the second support. Incertain embodiments, the first support and the second support may bedistinguished by the differential fluorescence of the first and secondsupports, and/or by differential scattering by first and second supportshaving different sizes.

In certain embodiments of the multiplex assay method above, theconjugative group linking the first fluorophore to the support may bethe same as or different from the conjugative group linking the secondfluorophore to the support. By way of non-limiting example, in oneembodiment the conjugative group may be different in order toselectively link one of the fluorophores with a support, while the otherfluorophore is linked to a different support or left freely suspended inthe test or reference sample. In other non-limiting exemplaryembodiments, the conjugative group is the same for both the first andsecond fluorophores, for example when the fluorophores are linked to thesame or different support temporally and/or spatially independently ofone another, when the fluorophores are linked to the same support,and/or when the fluorophores have sufficiently distinct fluorescencesignals that separation of the fluorophores during the fluorescencemeasurement is not necessary.

In various embodiments of the multiplex assay method above, the firstfluorophore and the second fluorophore may be linked to the samesupport, the same type of support, a different support, or a differenttype of support.

By way of example, a coupled assay may include at least one first enzymethat generates a first reaction product from a first enzymatic reactionon a first substrate, and at least one second enzyme that uses the firstreaction product generated from the first enzymatic reaction to producea fluorescence signal by reacting with and releasing the enzyme-reactivequenching group on the latent fluorophore. In certain embodiments, thefluorescence signal may be approximately proportional to theconcentration of the first substrate.

According to various exemplary and non-limiting embodiments of thedisclosure, the at least one first enzyme may be chosen from protease,phosphatase, and dehydrogenase. In at least one exemplary embodiment,the first enzyme is a dehydrogenase, and the first enzymatic reaction isa dehydrogenation reaction. In certain embodiments, the first substrateis phenylalanine, the first enzyme is phenylalanine dehydrogenase, andthe first reaction products are NADH (or NADPH)+phenylpyruvate +NH₃+H.The first substrate, first enzyme, and first reaction products for otherexemplary embodiments are shown in Table 1 below.

TABLE 1 First Substrate First Enzyme First Reaction Product CholesterolCholesterol NADH + cholest-4-en-3- dehydrogenase one + H⁺ LactateLactate dehydrogenase NADH + pyruvate + H⁺ Leucine Leucine dehydrogenaseNADH + 4-methyl-2- oxopentanoate + NH₃ + H⁺ D-glucose Glucosedehydrogenase NADH + D-glucono-1,5- lactone + H⁺ Ethanol Alcoholdehydrogenase NADH + acetaldehyde

Other non-limiting examples of enzymes contemplated according to variousembodiments of the disclosure may use modification of an enzyme-reactivegroup to achieve specificity for the enzyme of interest, for examplealkaline phosphatase using a rhodamine fluorophore (Levine, M. N. et al,Analytical Biochemistry 418 (2011), 247-252); esterase (Levine, M. N. etal, Molecules (2008), 13, 204-211); and serine protease using abis(N-benzyloxycarbonyl-L-argininamido)Rhodamine substrate (Leytus, S.P. et al, Biochem. J. (1983), 209, 299-307).

According to various exemplary embodiments, the at least one secondenzyme may be a diaphorase. As used here, the term “diaphorase” isintended to encompass several different enzyme classes, some of whichhave different substrate specificities for NADH or NADPH. By way ofexample only, the diaphorase may be chosen from the enzyme classes EC1.8.1.4-dihydrolipoyl dehydrogenase, EC 1.6.5.2-NAD(P)H dehydrogenase(quinone or DT-diaphorase), EC 1.6.99.1-NADPH dehydrogenase, EC1.6.99.3-NADH dehydrogenase, and mixtures thereof, all of which maycatalyze oxidation/reduction reactions. In at least one exemplaryembodiment, the diaphorase may be chosen from dihydrolipoyldehydrogenase and NAD(P)H dehydrogenase.

As shown in FIG. 1, an exemplary coupled assay for measuring the amountof an analyte in a sample may include a dehydrogenase and a diaphorase.In this exemplary embodiment, the dehydrogenase reacts with the analytein the sample to produce an oxidized analyte and NADH in a dehydrogenasereaction. The diaphorase uses the NADH created by the dehydrogenasereaction to react with and release the enzyme-reactive quenching groupfrom the latent fluorophore, thus revealing a fluorescent compound.

In various embodiments, the concentrations of dehydrogenase anddiaphorase may be adjusted such that the oxidation of the analyte is therate-limiting reaction.

As shown in FIG. 2, an exemplary coupled assay for measuring the amountof phenylalanine in a sample may include a phenylalanine dehydrogenase(PheDH) and a diaphorase. In this embodiment, the PheDH reacts withphenylalanine in the sample to produce phenylpyruvate, NH₃ and NADH in adehydrogenase reaction. The diaphorase uses the NADH created by thedehydrogenase reaction to react with and release the enzyme-reactivequenching group from the latent fluorophore, thus revealing afluorescent compound.

In various embodiments, the concentrations of PheDH and diaphorase maybe adjusted such that the oxidative deamination of phenylalanine is therate-limiting reaction.

In various exemplary embodiments, a direct assay may include at leastone enzyme that acts upon a substrate to produce a fluorescence signalby reacting with and releasing the enzyme-reactive quenching group onthe latent fluorophore. In at least one exemplary embodiment, the enzymemay be pyruvate oxidase and the substrate may be pyruvate.

As shown in FIG. 3, an exemplary direct assay for measuring the amountof pyruvate in a sample includes a pyruvate oxidase. In this exemplaryembodiment, the pyruvate oxidase reacts with the pyruvate in the sampleto produce acetate and CO₂, and also reacts with and releases a quinoneenzyme-reactive quenching group from the latent fluorophore, thusrevealing a fluorescent compound.

Methods of Detecting and Measuring Analytes in a Sample

Enzyme-reactive latent fluorophores linked to microspheres preparedaccording to various embodiments described herein may, in exemplaryembodiments, be used to detect the presence and/or concentration ofcertain analytes in a sample. The analyte may, for example, be detectedby utilizing a direct or coupled assay.

Exemplary methods for detecting and measuring an analyte in a sampleinclude one or more steps chosen from:

a. preparing a fluorophore composition comprising:

-   -   i. at least one enzyme-reactive latent fluorophore comprising at        least one enzyme-reactive quenching group and at least one        conjugative group, and    -   ii. a support connectable to the latent fluorophore by at least        one conjugative group;

b. providing a test sample to be analyzed and a reference sample to beanalyzed;

c. contacting the test sample with the latent fluorophore composition,at least one first unquenching enzyme capable of releasing theenzyme-reactive quenching group from the latent fluorophore, and atleast one second enzyme capable of reacting with the analyte;

d. contacting the reference sample with the latent fluorophorecomposition and the at least one first unquenching enzyme;

e. measuring the fluorescence signal of the test sample and thefluorescence signal of the reference sample; and

f. comparing the fluorescence signal of the test sample with thefluorescence signal of the reference sample.

In various embodiments of the method of measuring the activity of anenzyme above, the first unquenching enzyme that unlocks the latentfluorophore added to the test sample is the same as or different fromthe second enzyme capable of reacting with the analyte. When the firstunquenching enzyme is the same as the second enzyme, this may bereferred to as a direct assay. When the first unquenching enzyme isdifferent from the second enzyme, this may be referred to as an enzymecascade or coupled assay, wherein the first enzyme is considereddownstream of the second enzyme because the first enzyme requires aby-product of the reaction between the second enzyme and the analyte inorder to unquench the quenching group.

By way of non-limiting example, an assay, e.g. a coupled assay, may beused to measure the presence and/or concentration of phenylalanine inwhole blood. In at least certain exemplary embodiments, an assay, e.g. acoupled assay, can be used to measure the presence and/or concentrationof any analyte in any solution, e.g. those that have a specificdehydrogenase that generates NADH or NADPH in a solution, for examplewhole blood. In various embodiments, the assay can be used to measurethe presence and/or concentration of a metabolite, for example a humanmetabolite or an animal metabolite, in a bodily fluid, for exampleblood, urine, saliva, bile, spinal fluid, gastric juices, mucus, tears,amniotic fluid, semen, sweat, lymph fluids, and the like.

FIG. 4 a is an exemplary reaction schematic showing a latent fluorescentcompound revealed by a trimethyl lock reaction. In this exemplaryembodiment, the latent fluorophore having an enzyme-reactive quenchinggroup is connected to a microsphere with a linking group including apyrrole-2,5-dione bioconjugate and a PEG linker. In the presence of asuitable enzyme, for example diaphorase, and any cofactors, for exampleNADH, the enzyme-reactive quenching group is released from the latentfluorophore via a trimethyl lock reaction. The fluorophore connected tothe microsphere is now fluorescent.

FIG. 4 b is an exemplary reaction schematic showing a latent fluorescentcompound revealed by a trimethyl lock reaction and connected to amicrosphere with a biotin-streptavidin linkage. In this exemplaryembodiment, the latent fluorophore having an enzyme-reactive quenchinggroup and a biotin group is connected to a microsphere coated withstreptavidin. In the presence of a suitable enzyme, for examplediaphorase, and any cofactors, for example NADH, the enzyme-reactivequenching group is released from the latent fluorophore via a trimethyllock reaction. The fluorophore connected to the microsphere is nowfluorescent.

FIG. 5 is an exemplary reaction schematic showing a latent fluorescentcompound revealed by a double trimethyl lock reaction. In this exemplaryembodiment, the latent fluorophore has two enzyme-reactive quenchinggroups, and is connected to a microsphere with a linking group includinga pyrrole-2,5-dione bioconjugate and a PEG linker. In the presence of anenzyme, for example diaphorase, and any necessary cofactor, for exampleNADH, the enzyme releases one or both of the enzyme-reactive groups. Incertain embodiments, having two enzyme-reactive groups can help increaseor maximize the difference in fluorescence intensity upon reaction withthe enzyme.

In various embodiments, the microspheres may be used to isolate thefluorophores from the sample solution prior to the fluorescencemeasurement. In at least one embodiment, fluorophores connected tomagnetic microspheres may be collected using a magnet, and the otherreactants, compounds, and materials in the sample may be washed away. Incertain embodiments, the isolated magnetic microspheres may be suspendedin a suitable buffer, for example phosphate-buffered saline. Thefluorescence of the fluorophores linked to the magnetic microspheres canthen be then measured.

The fluorescence of the active fluorophore may be measured by any knownprocess, including but not limited to bulk fluorescence, flow cytometry,or spatially modulated fluorescence detection technology.

In certain exemplary and non-limiting embodiments according to thedisclosure, the fluorescence signal may be measured using bulkfluorescence measurements. In certain embodiments, fluorescence in thesample may be induced by directing light of an appropriate wavelengthregion into the sample that contains the fluorophores. Fluorescent lightof longer wavelengths than the excitation light may be collected anddetected from the same sample region. To block out unwanted excitationlight from the detection path, the optical axis of excitation anddetection path may be arranged perpendicularly or in opposing direction(so called epi-detection). Additionally, excitation and background lightcan be separated and filtered with wavelength selective (dichroic)mirrors and filters, for example bandpass filters.

In yet further exemplary embodiments, the fluorescence signal may bemeasured using flow cytometry, the principles of which are well known,and thus one of skill in the art would be able to determine theappropriate procedures and/or parameters for use according to variousembodiments of the disclosure.

In flow cytometry, the same measurement principles as in bulkfluorescence detection are applied. However, rather than measuring bulkfluorescence, the fluorescence intensity of particles is detected in aflow cytometer. The excitation and detection region of a flow cytometerare placed in the path of particles that are transported through afluidic system. The excitation/detection region covers usually thelateral width of the flow channel and expands some tens of micrometersalong the flow direction. Typical particle sizes detected in a flowcytometer generally range from 100 s nm to 10 s of μm. The size of theexcitation/detection region is limited in flow direction to ensure thatideally only a single particle is present in this region at any giventime. Commonly, flow cytometers can excite and detect in severalwavelength regions, including scattered light at the excitationwavelength. The subsequent (fluorescence) intensity measurement ofhundreds, thousands or even ten thousands of particles contains thestatistical information of the basic population.

In flow cytometry it is common practice to measure particle sizes byforward scattering signals. This measurement requires a precisealignment of the excitation laser and the detector by a light block.This block prevents laser light from entering the detector when noparticle is present. A particle with a refractive index (“RI”) differentfrom the suspension solution may scatter light within the excitationspot of the laser. This scattered light is detected by the scatterdetector, because it passes by the sides of the light block. Theintensity of the scattered light depends on a variety of particleproperties, including RI, size, and scatter center distribution. In aforward scattering direction, the amount of scattered light is usually ameasure of particle size, as the RI of bioparticles does not differ frombioparticle type to bioparticle type enough to provide relevantbioparticle information while size (shape, orientation) dominates thescatter properties of the particle.

In yet further exemplary embodiments, the fluorescence signal may bemeasured using spatially modulated fluorescence detection technology,which uses fluorescence detection comparable to a flow cytometer.However, the spatial modulation technique uses a largeexcitation/detection area (ca. 0.1×1 mm) to increase the total flux offluorescence light that originates from a particle. This configurationgenerates a time-dependent signal as a continuously fluorescing particletraverses a predefined pattern for optical transmission. Correlating thedetected signal with the known pattern achieves high discrimination ofthe particle signal from background noise and simplifies optical systemalignment. At the same time, multiple particles that simultaneouslyoccur in the detection region can be differentiated computationally bythe correlation of the signal sequences and the known transmission maskpattern.

FIG. 6 is a flowchart showing the steps for an exemplary dehydrogenasedifferential assay of a sample, for example blood. The sample is dividedbetween a test sample and a reference sample. The test sample containsthe analyte-specific enzyme, for example dehydrogenase, the diaphorase,the latent fluorophore, plus any necessary cofactors, for example NAD+.The reference sample contains all of the components in the test sampleexcept for the analyte-specific enzyme. The fluorescence signals fromthe test sample and the reference sample are measured, and thefluorescence signal of the reference sample is subtracted from that ofthe test sample to yield a differential measurement.

In certain embodiments, differential measurements may be desired, e.g.to increase the reliability of the assay. For example, some individualsmay have varying levels of other analytes in their blood, which canresult in different background signals that are independent of enzymaticaction on the desired analyte.

FIG. 7 is a flowchart showing the steps for an exemplary phenylalaninedehydrogenase differential assay of blood. The sample is divided betweena test sample and a reference sample. The test sample containsphenylalanine dehydrogenase (PheDH), the diaphorase, the latentfluorophore, plus any necessary cofactors, for example NAD+. Thereference sample contains all of the components in the test sampleexcept for the PheDH. The fluorescence signals from the test sample andthe reference sample are measured, and the fluorescence signal of thereference sample is subtracted from that of the test sample to yield adifferential measurement.

In certain embodiments, the latent fluorophore including a suitablelinking group, for example biotin, can be used in solution and bound tothe microsphere coated with a suitable functional group, for examplestreptavidin, after the enzyme assay is performed. FIG. 8 a is areaction schematic showing an exemplary embodiment of a latentfluorescent compound revealed by a trimethyl lock reaction andsubsequently linked to a streptavidin-coated microsphere. The enzymeassay is performed as described above using latent fluorophoresincluding a biotin group. Streptavidin-coated microspheres aresubsequently introduced into the test sample and reference sample. Thebiotin binds to the streptavidin, which connects the fluorophore to themicrosphere. The microsphere can then be used to isolate the fluorophorefrom the samples as described above.

In further exemplary embodiments according to the disclosure, amultiplex array can be used to examine the activity of multiple enzymesat the same time by using different enzyme-reactive quenching groups anda different fluorophore for each enzymatic reaction. The fluorescence ofthe different fluorophores connected to different microspheres can beanalyzed using flow cytometry or spatially modulated fluorescencedetection technology. Suitable enzymes include, but are not limited to,proteases, phosphatases, and dehydrogenases.

FIG. 9 a is a flowchart showing the steps for a general enzymaticmultiplexed differential assay of a sample using fluorophores havingdifferent emission properties, according to an exemplary embodiment ofthe disclosure. Although three different enzymes are used in theexemplary embodiment shown in FIG. 9 a, a greater or fewer number ofenzymes may be used. The sample is divided between a test sample and areference sample. The test sample contains three different enzymes,three different latent fluorophores, plus any necessary cofactors andcoupling enzymes, for example diaphorase. The reference sample containsall of the components in the test sample except for the three differentenzymes. The three different fluorophores have different enzyme-reactivequenching groups and different emission properties, for example maxima.The fluorescence signals from the test sample and the reference sampleare measured, and the fluorescence signals of the reference sample aresubtracted from those of the test sample to yield differentialmeasurements. In certain embodiments, the microspheres are magnetic.

FIG. 9 b is a flowchart showing the steps for a general enzymaticmultiplexed differential assay of a sample using fluorescentmicrospheres having different emission properties according to anexemplary embodiment of the disclosure. Although three different enzymesare used in the exemplary embodiment shown in FIG. 9 b, a greater orfewer number of enzymes may be used. The sample is divided between atest sample and a reference sample. The test sample contains threedifferent enzymes, three different latent fluorophores, plus anynecessary cofactors and coupling enzymes, for example diaphorase. Thereference sample contains all of the components in the test sampleexcept for the three different enzymes. The three different fluorophoreshave different enzyme-reactive quenching groups and the same emissionproperties. Each of the three different latent fluorophores is coupledto a microsphere having a specific fluorescent property, i.e., latentfluorophore 1 is coupled to microsphere type 1, latent fluorophore 2 iscoupled to microsphere type 2, and latent fluorophore 3 is coupled tomicrosphere type 3, where microsphere types 1-3 have differentfluorescent properties, for example emission maxima. Although the threelatent fluorophores have the same fluorescence properties, the activityof the three different enzymes may be monitored by separating themicrobeads having specific fluorescent properties, for example by flowcytometry. The fluorescence signals from the fluorophores and/ormicrospheres of the test sample and the reference sample are measured,and the fluorescence signals of the reference sample are subtracted fromthose of the test sample to yield differential measurements. In anotherembodiment, the three latent fluorophores have different emissionproperties. In certain embodiments, all or some of the microspheres aremagnetic.

FIG. 9 c is a flowchart showing the steps for a general enzymaticmultiplexed differential assay of a sample using fluorescentmicrospheres having different sizes, according to an exemplaryembodiment of the disclosure. Although three different enzymes are usedin the exemplary embodiment shown in FIG. 9 c, a greater or fewer numberof enzymes may be used. The sample is divided between a test sample anda reference sample. The test sample contains three different enzymes,three different latent fluorophores, plus any necessary cofactors andcoupling enzymes, for example diaphorase. The reference sample containsall of the components in the test sample except for the three differentenzymes. The three different fluorophores have different enzyme-reactivequenching groups and the same emission properties. Each of the threedifferent latent fluorophores is coupled to a microsphere having aspecific particle size, i.e., latent fluorophore 1 is coupled tomicrosphere type 1, latent fluorophore 2 is coupled to microsphere type2, and latent fluorophore 3 is coupled to microsphere type 3, wheremicrosphere types 1-3 have different sizes. The microspheres may bemagnetic and/or fluorescent, with the same or different fluorescentproperties. The difference in particle sizes between the differentmicrospheres causes different scattering signals from each size ofmicrosphere. Although the three latent fluorophores have the samefluorescence properties, the activity of the three different enzymes maybe monitored by distinguishing the microspheres by size based on thedifferent scattering properties, for example using flow cytometry. Thefluorescence signals and scattering signals from the fluorophores and/ormicrospheres of the test sample and the reference sample are measured,and the fluorescence signals of the reference sample are subtracted fromthose of the test sample to yield differential measurements. In anotherembodiment, the three latent fluorophores have different emissionproperties.

In at least certain exemplary embodiments, the enzymatic multiplexeddifferential assays shown in FIGS. 9 a-9 c may be applied tosimultaneously screen compounds on multiple enzyme targets in amultiplexed differential screening assay. For example, a multiplexeddifferential screening assay may be used to determine which, if any, ofa number of enzymes shows activity in the presence of a test compound.

FIG. 10 a is a flowchart showing the steps for a general enzymaticmultiplexed differential screening assay of a sample using fluorophoreshaving different emission properties, according to an exemplaryembodiment of the disclosure. Although three different enzymes are usedin the exemplary embodiment shown in FIG. 10 a, a greater or fewernumber of enzymes may be used. The sample is divided between a testsample and a reference sample. The test sample contains three differentenzymes, three different latent fluorophores, a test compound, plus anynecessary cofactors and coupling enzymes, for example diaphorase. Thereference sample contains all of the components in the test sampleexcept for the test compound. The three different fluorophores havedifferent enzyme-reactive quenching groups and different emissionproperties, for example maxima. Although the three latent fluorophoreshave the same fluorescence properties, the activity of the threedifferent enzymes may be monitored by separating the microbeads havingspecific fluorescent properties, for example by flow cytometry. Thefluorescence signals from the test sample and the reference sample aremeasured, and the fluorescence signals of the reference sample aresubtracted from those of the test sample to yield differentialmeasurements. In certain embodiments, the microspheres are magnetic

FIG. 10 b is a flowchart showing the steps for a general enzymaticmultiplexed differential screening assay of a sample using fluorescentmicrospheres having different emission properties, according to anexemplary embodiment of the disclosure. Although three different enzymesare used in the exemplary embodiment shown in FIG. 10 b, a greater orfewer number of enzymes may be used. The sample is divided between atest sample and a reference sample. The test sample contains threedifferent enzymes, three different latent fluorophores, a test compound,plus any necessary cofactors and coupling enzymes, for examplediaphorase. The reference sample contains all of the components in thetest sample except for the test compound. The three differentfluorophores have different enzyme-reactive quenching groups and thesame emission properties. Each of the three different latentfluorophores is coupled to a microsphere having a specific fluorescentproperty, i.e., latent fluorophore 1 is coupled to microsphere type 1,latent fluorophore 2 is coupled to microsphere type 2, and latentfluorophore 3 is coupled to microsphere type 3, where microsphere types1-3 have different fluorescent properties, for example emission maxima.Although the three latent fluorophores have the same fluorescenceproperties, the activity of the three different enzymes may be monitoredby separating the microbeads having specific fluorescent properties, forexample by flow cytometry. The fluorescence signals from thefluorophores and/or microspheres of the test sample and the referencesample are measured, and the fluorescence signals of the referencesample are subtracted from those of the test sample to yielddifferential measurements. In another embodiment, the three latentfluorophores have different emission properties. In certain embodiments,all or some of the microspheres are magnetic.

FIG. 10 c is a flowchart showing the steps for a general enzymaticmultiplexed differential screening assay of a sample using fluorescentmicrospheres having different particle sizes, according to an exemplaryembodiment of the disclosure. Although three different enzymes are usedin the exemplary embodiment shown in FIG. 10 c, a greater or fewernumber of enzymes may be used. The sample is divided between a testsample and a reference sample. The test sample contains three differentenzymes, three different latent fluorophores, a test compound, plus anynecessary cofactors and coupling enzymes, for example diaphorase. Thereference sample contains all of the components in the test sampleexcept for the test compound. The three different fluorophores havedifferent enzyme-reactive quenching groups and the same emissionproperties. Each of the three different latent fluorophores is coupledto a microsphere having a specific particle size, i.e., latentfluorophore 1 is coupled to microsphere type 1, latent fluorophore 2 iscoupled to microsphere type 2, and latent fluorophore 3 is coupled tomicrosphere type 3, where microsphere types 1-3 have different sizes.The microspheres may be magnetic and/or fluorescent, with the same ordifferent fluorescent properties. The difference in particle sizesbetween the different microspheres causes different scattering signalsfrom each size of microsphere. Although the three latent fluorophoreshave the same fluorescence properties, the activity of the threedifferent enzymes may be monitored by distinguishing the microspheres bysize based on the different scattering properties, for example duringflow cytometry. The fluorescence signals from the fluorophores and/ormicrospheres of the test sample and the reference sample are measured,and the fluorescence signals of the reference sample are subtracted fromthose of the test sample to yield differential measurements. In anotherembodiment, the three latent fluorophores have different emissionproperties. In certain embodiments, all or some of the microspheres aremagnetic.

Further exemplary embodiments of the disclosure relates to kitscomprising a composition, where the composition comprises at least onelatent fluorophore comprising at least one enzyme-reactive quenchinggroup and at least one conjugative group, and at least one supportconnectible to the at least one latent fluorophore via at least oneconjugative group, as described herein. These kits may include, forexample, at-home testing kits for analyzing phenylalanine levels inblood.

As used herein, the terms “a”, “an”, and “the” are intended to encompassthe plural as well as the singular. In other words, for ease ofreference only, the terms “a” or “an” or “the” may be used herein, suchas “a support”, “an enzyme”, “the microsphere”, etc., but are intended,unless explicitly indicated to the contrary, to mean “at least one,”such as “at least one support”, “at least one enzyme”, “the at least onemicrosphere”, etc. This is true even if the term “at least one” is usedin one instance, and “a” or “an” or “the” is used in another instance,e.g. in the same paragraph or section. Furthermore, as used herein, thephrase “at least one” means one or more, and thus includes individualcomponents as well as mixtures/combinations.

The term “comprising” (and its grammatical variations) as used herein isused in the inclusive sense of “having” or “including,” with which itmay be used interchangeably. These terms are not to be construed asbeing used in the exclusive sense of “consisting only of” unlessexplicitly so stated.

Other than where expressly indicated, all numbers expressing quantitiesof ingredients and/or reaction conditions are to be understood as beingmodified in all instances by the term “about.” This includes terms suchas “all” or “none” and variants thereof. As used herein, the modifier“about” means within the limits that one of skill in the art wouldexpect with regard to the particular quantity defined; this may be, forexample, in various embodiments, ±10% of the indicated number, ±5% ofthe indicated number, ±2% of the indicated number, ±1% of the indicatednumber, ±0.5% of the indicated number, or ±0.1% of the indicated number.

Additionally, where ranges are given, it is understood that theendpoints of the range define additional embodiments, and that subrangesincluding those not expressly recited are also intended to includeadditional embodiments.

As used herein, “formed from,” “generated by,” and variations thereof,mean obtained from chemical reaction of, wherein “chemical reaction,”includes spontaneous chemical reactions and induced chemical reactions.As used herein, the phrases “formed from” and “generated by” are openended and do not limit the components of the composition to thoselisted.

The compositions and methods according to the present disclosure cancomprise, consist of, or consist essentially of the elements andlimitations described herein, as well as any additional or optionalingredients, components, or limitations described herein or otherwiseknown in the art.

It should be understood that, unless explicitly stated otherwise, thesteps of various methods described herein may be performed in any order,and not all steps must be performed, yet the methods are still intendedto be within the scope of the disclosure.

It will be appreciated that variants of the above-disclosed and otherfeatures and functions, or alternatives thereof, may be combined intomany other different systems or applications. Various presentlyunforeseen or unanticipated alternatives, modifications, variations, orimprovements therein may be subsequently made by those skilled in theart which are also intended to be encompassed by the following claims.

What is claimed is:
 1. A composition comprising: a. at least one latentfluorophore comprising: i. at least one enzyme-reactive quenching group,and ii. at least one conjugative group; and b. at least one supportconnectable to the latent fluorophore.
 2. The composition of claim 1,wherein the at least one support is connected to the latent fluorophoreby the at least one conjugative group.
 3. The composition of claim 1,wherein the at least one support is chosen from microspheres.
 4. Thecomposition of claim 3, wherein the at least one support is magnetic. 5.The composition of claim 1, wherein the at least one conjugative groupis chosen from bioconjugative groups, PEG linkers, biotin groups, andcombinations thereof.
 6. The composition of claim 1, where the at leastone latent fluorophore is chosen from trimethyl lock fluorophores. 7.The composition of claim 6, wherein the trimethyl lock fluorophore ischosen from compounds of Chemical Formula 1, Chemical Formula 2,Chemical Formula 3, and combinations thereof:


8. The composition of claim 1, wherein the at least one latentfluorophore comprises at least one diaphorase-reactive quenching groupthat is releasable from the latent fluorophore by a diaphorase.
 9. Amethod of detecting and/or measuring the concentration of an analyte ina sample, the method comprising: a. providing a composition comprising:i. at least one latent fluorophore comprising at least oneenzyme-reactive quenching group and at least one conjugative group, andii. at least one support connectable to the at least one latentfluorophore by the at least one conjugative group; b. providing a testsample and a reference sample; c. contacting the test sample with thecomposition, at least one first unquenching enzyme capable of releasingthe enzyme-reactive quenching group from the latent fluorophore, and atleast one second enzyme capable of reacting with the analyte; and d.contacting the reference sample with the latent fluorophore compositionand the at least one first unquenching enzyme.
 10. The method of claim9, further comprising: e. measuring the fluorescence signal of the testsample and the fluorescence signal of the reference sample.
 11. Themethod of claim 10, further comprising: f. comparing the fluorescencesignal of the test sample with the fluorescence signal of the referencesample.
 12. The method of claim 11, further comprising: g. connectingthe latent fluorophore to the support, and h. isolating the latentfluorophore composition from the sample.
 13. The method of claim 9,wherein the at least one support is chosen from microspheres.
 14. Themethod of claim 13, wherein the at least one support is magnetic. 15.The method of claim 9, wherein the at least one first unquenching enzymeand the at least one second enzyme are the same.
 16. The method of claim9, wherein the at least one second enzyme is chosen from diaphorase,esterase, phosphatase, pyruvate oxidase, and combinations thereof. 17.The method of claim 9, wherein the at least one first unquenching enzymeis capable of generating NADH or NADPH when reacted with the analyte.18. The method of claim 9, wherein the at least one first enzyme ischosen from a dehydrogenase, a protease, a phosphatase, and combinationsthereof.
 19. The method of claim 9, wherein the at least one conjugativegroup is chosen from bioconjugative groups, PEG linkers, biotin groups,and combinations thereof.
 20. The method of claim 9, wherein the atleast one latent fluorophore is chosen from trimethyl lock fluorophores.21. The method of claim 20, wherein the trimethyl lock fluorophore ischosen from compounds of Chemical Formula 1, Chemical Formula 2,Chemical Formula 3, and combinations thereof:


22. A method of measuring the activities of at least two enzymes in asample, the method comprising: a. providing a first fluorophorecomposition comprising: i. at least one first enzyme-reactive latentfluorophore comprising at least one first enzyme-reactive quenchinggroup and at least one conjugative group, and ii. at least one supportconnectable to the at least one first latent fluorophore by at least oneconjugative group; b. providing a second fluorophore compositioncomprising: i. at least one second enzyme-reactive latent fluorophorecomprising at least one second enzyme-reactive quenching group and atleast one conjugative group, wherein the at least one secondenzyme-reactive latent fluorophore is different from said firstenzyme-reactive latent fluorophore in said first fluorophorecomposition, and ii. at least one support connectable to the at leastone first latent fluorophore by at least one conjugative group; c.providing a test sample to be analyzed and a reference sample to beanalyzed; d. contacting the test sample with the first and second latentfluorophore compositions, at least one first unquenching enzyme capableof releasing the enzyme-reactive quenching group from the first latentfluorophore, and at least one second unquenching enzyme capable ofreleasing the enzyme-reactive quenching group from the second latentfluorophore; e. contacting the reference sample with the first andsecond latent fluorophore compositions.
 23. The method of claim 22,further comprising: f. measuring the fluorescence signal of the testsample and the fluorescence signal of the reference sample.
 24. Themethod of claim 23, further comprising: g. comparing the fluorescencesignal of the test sample with the fluorescence signal of the referencesample.
 25. The method of claim 24, further comprising: h.distinguishing the fluorescence signal of the at least one first enzymefrom the fluorescence signal of the at least one second enzyme.